Nanocurcumin-mediated down-regulation of telomerase via stimulating TGF beta 1 signaling pathway in hepatocellular carcinoma cells

Background: Curcumin, extracted from turmeric, represents enormous potential to serve as an anticancer agent. Telomerase is viewed as a prominent molecular target of curcumin, and transforming growth factor- beta 1 [TGF beta 1] has proven to be a major inhibitory signaling pathway for telomerase activity. In the current study, we aimed to explore suppressive effects of nanocurcumin on telomerase expression through TGF beta 1 pathway in a hepatocellular carcinoma cell line [Huh7]

Methods: MTT assay was used to determine the effect of nonocurcumin on viability of Huh7 cells. RT-PCR was used to analyze the gene expression patterns

Results: MTT assay revealed that nanocurcumin acts in a dose- and time-dependent manner to diminish the cell viability. RT-PCR analysis indicated that nanocurcumin results in augmentation of TGF beta 1 72 hours post treatment and leads to the reduction of telomerase expression 48 and 72 hours post exposure. Also, up-regulation of Smad3 and E2F1 and down-regulation of Smad7 confirmed the effect of nanocurcumin on intermediate components of TGF beta 1 pathway. Furthermore, transfection of the proximal promoter of telomerase triggered a significant reduction in luciferase activity

Conclusion: The data from the present study lead us to develop a deeper understanding of the mechanisms underlying nanocurcumin-mediated regulation of telomerase expression, thereby presenting a new perspective to the landscape of using nanocurcumin as a cancer-oriented therapeutic agent

Functional surface display of laccase in a phenol-inducible bacterial circuit for bioremediation purposes

Background: Phenolic compounds, which are produced routinely by industrial and urban activities, possess dangers to live organisms and environment. Laccases are oxidoreductase enzymes with the ability of remediating a wide variety of phenolic compounds to more benign molecules. The purpose of the present research is surface display of a laccase enzyme with adhesin involved in diffuse adhesion [AIDA-I] autotransporter system on the surface of Escherichia coli cells for bioremediation of phenolic compounds

Methods: The expression of laccase was regulated by a phenol-responsive promoter [a 54 promoter]. The constitutively-expressed CapR transcription activator was able to induce laccase expression in the presence of phenolic compounds

Results: Western blot analysis showed the expression and correct transfer of the enzyme to the outer membrane of E. coli cells in the presence of phenol. Activity assay confirmed the correct folding of the enzyme after translocation through the autotransporter system. HPLC analysis of residual phenol in culture medium showed a significant reduction of phenol concentration in the presence of cells displaying laccase on the surface

Conclusion: Our findings confirm that autodisplay enables functional surface display of laccase for direct substrate-enzyme availability by overcoming membrane hindrance

Decrease in cavity size and oligodendrocyte cell death using Neurosphere-Derived oligodendrocyte-like cells in spinal cord contusion model

Background: Oligodendrocyte cell death is among the important features of spinal cord injury, which appears within 15 min and occurs intensely for 4 h after injury, in the rat spinal contusion model. Accordingly, the number of oligodendrocytes progressively reduced within 24 h after injury. Administration of oligodendrocyte-like cells [OLCs] into the lesion area is one of the approaches to counterbalance this condition

Methods: Bone marrow stromal cells were transdifferentiated into neurospheres and then into neural stem cells and later were differentiated into OLCs using triiodothyronine and transplanted into the spinal cord contusion rats. The postinjury functional recovery was explored and compared with the control group using Basso-Beattie-Bresnahan and narrow beam behavioral tests. At the end of 12th week, spinal cord segments T12-L1 were histomorphologically studied by immunohistochemistry

Results: Motor improvement was more obvious during 2nd to 4th weeks and got less prominent during 4th to 12th weeks. Histomorphometric findings indicated that cavity formation decreased in epicenter of transplantation area in experimental groups in comparison with the control groups

Conclusion: The findings obtained in the present study showed that OLC therapy is a potential approach in the treatment of spinal cord traumatic injuries

Preparation and evaluation of rifampicin and co-trimoxazoleloaded nanocarrier against brucella melitensis infection

Background: Brucellosis or Malta fever is a contagious infection common between human and domestic animals. Many antibiotics are used for brucellosis treatment, but they are not efficient and put heavy burden on society. Co-trimoxazole and rifampicin are two candidates for brucellosis treatment. In this study, we aimed to enhance the efficacy of these antibiotics using designed nanoparticles

Methods: Different concentrations of cotrimoxazole and rifampicin were used for loading onto a nanostructure of synthesized monomethoxy poly[ethylene glycol]-oleate [mPEG-OA]. The solubility, cytotoxicity, and efficacy of these nano-packed antibiotics on Brucella-infected murine phagocytic cells were examined, as compared with free antibiotics. Then the release nanoparticles was increased approximately 3.5 and 1.5fold, respectively, which is considerable in comparison with free insoluble ones

Results: Despite acceptable loading percentage, the application of co-trimoxazole-loaded nanoparticle on Brucella-infected J774A.1 murine macrophage-like cells did not lead to reduction in the number of bacteria; however, the efficacy of rifampicin on Brucella-infected murine phagocytic cells enhanced

Conclusion: In the current study, the efficacy of rifampicin on reducing the number of Brucella melitensis increased by the novel synthesized nanostructure. In contrast, since co-trimoxazole efficacy did not enhance by loading onto nanoparticles, the co-trimoxazole inefficiency is most likely not due to its low penetration or insolubility, and probably there are other factors that remain to be clarified in the future investigations

[Study of loading, cytotoxicity, uptake, and release of curcumin from a novel gemini surfactant nanocarrier]

Objective: Numerous researches have been conducted to comprehend the anti-cancer effects of curcumin [Cu]. Although the anti-proliferative properties of Cu on cancerous cells is known, the clinical application of this gold substrate is limited. This limitation is mostly due to low solubility, inefficient bioavailability, rapid metabolism, and improper uptake. In this study, we have synthesized a novel biodegradable gemini surfactant [Gs], after which the curcumin [Cu] molecules were encapsulated within the polymer to overcome its physicochemical limitations

Methods: We prepared Gs-Cu nanoparticles by the nanoprecipitation method. Size and polydispersity index of the nanoparticles were determined by the dynamic light scattering [DLS] technique. The release profile of Cu from the polymer matrix was studied, and the MTT assay and cellular uptake of Gs-Cu on MDA-MB-231 cells were investigated in vitro

Results: The Gs polymer had the capability to form polymersomes in an aqueous solution; a narrow size distribution was obtained [PDI=0.3]. The encapsulation efficiency approximated 87%. We observed a sustained release profile due to incorporation of Cu into the polymer matrix. The Gs-Cu complex showed more cytotoxicity compared to free Cu because of the higher rate of cellular internalization

Conclusions: The data indicate that Gs polymersomes can be regarded as nanocarriers for hydrophobic curcumin molecules

In silico analysis of an envelope domain III-based multivalent fusion protein as a potential dengue vaccine candidate

PURPOSE: Dengue virus infection is now a global problem. Currently, there is no licensed vaccine or proven antiviral treatment against this virus. All four serotypes (1-4) of dengue virus can infect human. An effective dengue vaccine should be tetravalent to induce protective immune responses against all four serotypes. Most of dengue vaccine candidates are monovalent, or in the form of physically mixed multivalent formulations. Recently envelope protein domain III of virus is considered as a vaccine candidate, which plays critical roles in the most important viral activities. Development of a tetravalent protein subunit vaccine is very important for equal induction of immune system and prevention of unbalanced immunity. Here, we have presented and used a rational approach to design a tetravalent dengue vaccine candidate. MATERIALS AND METHODS: We designed a multi domain antigen by fusing four consensus domain III sequences together with appropriate hydrophobic linkers and used several types of bioinformatics software and neural networks to predict structural and immunological properties of the designed tetravalent antigen. RESULTS: We designed a tetravalent protein (EDIIIF) based on domain III of dengue virus envelope protein. According to the results of the bioinformatics analysis, the constructed models for EDIIIF protein were structurally stable and potentially immunogenic. CONCLUSION: The designed tetravalent protein can be considered as a potential dengue vaccine candidate. The presented approach can be used for rational design and in silico evaluation of chimeric dengue vaccine candidates.

[One-step synthesis of anionic PEG-citrate G2 dendrimer and evaluation of its cytotoxicity in two cell lines]

Objective: Dendrimers are three-dimensional nanostructures that have numerous applications in medicine, including drug delivery and imaging. Although anionic dendrimer polyethylene glycol-citrate has a high potential to increase solubility of waterinsoluble drugs and drug delivery, its multi-step synthesis procedure is time consuming. In addition, toxic substances such as dichloromethane are used in its synthesis procedure. In this study, we have developed a simple one-step synthesis method using green chemistry

Methods: We examined four different methods to improve the synthesis method of this dendrimer. Products were characterized by FTIR, LC-MS and DLS. Cytotoxicity was assessed by the XTT method

Results: We synthesized a G2 polyethylene glycol-citrate dendrimer in one-step without purifying G1. This process was chosen as a beneficial method for synthesis of the G2 dendrimer. When compared with previous methods, this procedure had higher efficiency and greatly reduced response. This procedure used nontoxic materials. XTT assay results showed that this dendrimer created by green chemistry had no cytotoxicity in Hela and Vero cells up to a concentration of 800 microM

Conclusion: One-step synthesis of anionic polyethylene glycol-citrate G2 dendrimer is a simple, beneficial production method. The dendrimer is biocompatible and can be used as a suitable carrier for drug delivery purposes

[Comparison of snRNA-U6 and microRNA-16 for identification of suitable endogenous control gene for microRNA gene expression analysis under dendrosomal curcumin treatment in hepatocellular carcinoma cell lines]

Objective: MicroRNAs [miRNAs] are single-stranded small RNAs 18-25 nucleotides in length that regulate gene expression through translational inhibition and mRNA cleavage. Aberrant expression of miRNAs contribute to several diseases. This has increased interest in profiling the expressions of these molecules. Real-time quantitative PCR [RQ-PCR] is a sensitive, quantitative technique for gene expression assessment. To correct for systematic variables such as the amount of starting template, RNA quality and enzymatic efficiencies, RQ-PCR data is commonly normalized to an endogenous control gene which is stably-expressed across the test sample set. To avoid occurring further error in the quantification of gene expression data, it is necessary that candidate endogenous controls be validated in the samples of interest. In this study the expression of miRNA-16 and small nuclear RNA [snRNA]-U6 in hepatocellular carcinoma [HCC] cell lines under dendrosomal curcumin treatment were evaluated to identify appropriate endogenous controls for dendrosomal curcumin-related miRNA expression assays

Methods: HCC cell lines were treated with dendrosomal curcumin. Dendrosomal curcumin entry into HepG2 and HuH-7 cells was assessed by fluorescent microscopy images. RNA was extracted and cDNA, after polyA polymerization, was synthesised. Then, we performed gene expression assays using RQ-PCR

Results: In this treatment condition, miRNA-16 for HepG2, snRNA-U6 and the combined miRNA-16 and snRNA-U6 for HuH-7 were suitable endogenous controls

Conclusion: These genes are appropriate endogenous controls for miRNA expression assays in HCC cell lines under treatment with dendrosomal curcumin. There are stable, non-significant expression changes of these genes

[Identification of novel targeting peptides for PC3 cells by the screening of a phage display peptide library]

Objective: Prostate cancer is the second cause of cancer-associated death in men. In recent years, targeted therapy for cancer has attracted the attention of researchers. Targeted therapy leads to a decrease in drug adverse effects. Studies indicate that targeting peptides for cancer cells represent valuable tools for diagnostics and therapeutics. Recently, phage display peptide libraries have been used to identify target peptides to a variety of cancer cells. In the current study, we aim to isolate peptides that target PC3 cells [human prostate adenocarcinoma cells]

Methods: Four rounds of subtractive panning on control cells that included 5637 [bladder], Huh-7 [liver], SW480 [colon], AGS [stomach] and human fibroblast normal in addition to four rounds of positive panning on PC3 [target cell] were performed. Polyclonal phage ELISA was used to evaluate the process of enrichment during biopanning. Subsequently, phage clones were randomly selected from titer plates, amplified by plaque-PCR, and their genomic DNA was sequenced. We conducted bioinformatic analysis for further characterization of the isolated peptides

Results: Several rounds of panning resulted in the enrichment of a number of peptides. The results of polyclonal phage ELISA indicated that the biopanning process was successful. In silico analysis showed the presence of several consensus amino acid motifs in the peptides

Conclusion: The peptides identified through biopanning can be considered as potential specific binders to PC3 cells. Peptides with specificity binding to target cells can be used for targeted gene and drug delivery to malignant tumor cells. Further analyses of these peptides are required to show their capacity for targeted delivery of various genes and drugs into prostate cancer cells

[Transduction of mesenchymal stem cells that express VEGF-A and pre-conditioning with SDF1alpha In order to increase survival for use in myocardial infarction treatment in a rat model]

Objective: The use of stem cells, particularly mesenchymal stem cells [MSCs], with genes and various growth factors as treatments for myocardial infarction and various other diseases is highly regarded. However these cells meet with inflammation and a hypoxic environment in the target tissue. Hence, treatment with factors that increase the resistance of these stem cells is of importance. Stem cells also can be used as carriers for gene therapy. The aim of the present research is to produce VEGF expressing MSCs. We investigate the effect of stromal derived factor 1 on MSC survival in order to use these cells in a future rat myocardial infarction model

Methods: MSCs were purified from young male rats by aspirating the cavity of femurs and tibias. After characterization, MSCs were transduced with VEGF using lipofectamine. Expression and function of VEGF was confirmed. Next, we treated MSCs with SDF1alpha at various time points. The effect of this chemokine was investigated using the LDH assay and by viable cell counts

Results: The experiments confirmed the production and function of VEGF by MSCs. The LDH levels decreased significantly in SDF1alpha treated MSCs. Cell viability increased significantly in the presence of this chemokine

Conclusion: Treatment of MSCs with the SDF1alpha chemokine has increased the survival of these cells. These MSCs are proper candidates for increasing angiogenesis and for further analysis in a rat model of myocardial infarction

[Design and synthesis of a novel dendrosome and a PEGylated PAMAM dendrimer nanocarrier to improve the anticancer effect of turmeric [curcuma longa] curcumin]

In recent decades, the anticancer effect of curcumin has been proven by several studies. Curcumin affects multiple cell signaling pathways and prevents cell proliferation, invasion, metastasis and angiogenesis. However, the aqueous solubility of curcumin and its bioavailability are very low which restricts its anticancer properties. In this research, we have synthesized a monomethoxy poly [ethylene glycol]-Oleate [mPEGOA] di-block copolymer and used a surface PEGylated poly [amidoamine] [PAMAM] dendrimer to improve bioavailability of curcumin in cancer cells. The critical micelle concentration [CMC] of mPEG-OA, drug loading efficiencies, and cytotoxicity in the human glioblastoma cell line [U87MG] of all the prepared nanodevices were thoroughly investigated. Atomic force microscopy [AFM] and dynamic light scattering [DLS] studies have shown that mPEG-OA have two common nanostructures, micelles and polymerosomes. mPEG-OA micelles had a very low CMC [0.03 g/l]. The IC50 of free curcumin [0.01 methanol solution] was 48 microM, curcumin-loaded mPEG-OA was 24 microM, and curcumin-loaded PAMAM dendrimer was 13 microM. Moreover, the PEGylated PAMAM was non-cytotoxic. The results indicated that by using these nanocarriers, the bioavailability of curcumin significantly increased compared to free curcumin. Overall, this research revealed that these curcumin nanocarriers could be considered as appropriate drug delivery systems for curcumin delivery in cancer cells

Cloning and sequence analysis of gene encoding OipA from Iranian patients with helicobacter pylori infection

Outer inflammatory protein A [OipA] is one of the important adhesins of H. pylori and a valuable candidate for vaccine development. Its gene is under "on-off" switch status which correlates with OipA protein expression. We aimed to obtain a recombinant OipA clone [with "on" status] from an Iranian clinical isolate. A clinical H. pylori-isolate demonstrating high expression for an outer membrane protein [OMP] with an apparent MW of 33-35 kDa was selected. oipA specific primer was designed according to oipA sequences from B8 strain. The purified PCR-product was sequenced and submitted to Gene Bank. The pET-28a plasmid and E. coli DH5alpha were used for cloning and transformation. The recombinant plasmid was transferred to E. coli BL21 [DE3]. Extracted proteins were purified and presence of OipA was confirmed by western blotting using both anti His-tag monoclonal antibody and anti-OipA specific antibody. The sequence of the oipA gene and the MW of the purified recombinant OipA protein consisted on 924 bp and 33-35 kDa, respectively. Its identity with other published oipA genes was 92-96%; highest identity was observed with that of a Mexican oipA clone, obtained from a H. pylori strain associated with severe symptoms. Recombinant oipA clone obtained in this work, may be a functional oipA gene with "on" status, associated with more severe outcomes of H, pylori infection

Construction of a minigenome rescue system for measles virus, AIK-c strain

In the recent decade, the reverse genetics method has been broadly used for rescue of negative-stranded RNA viruses from cDNA or viral minigenomes. This technique has been applied to study different steps in virus replication and virus-host interactions. Reverse genetics could also be implemented for design of new vaccines. The T7 RNA polymerase activity as well as virus [nucleocapsid protein] N, [phosphoprotein] P and [Large] L proteins are necessary to rescue the virus or viral minigenome. Measles virus is a negative-stranded non-segmented RNA virus. There are useful vaccine strains to prevent measles disease. Here, we describe the construction of a new helper cell line for rescue of measles virus minigenome. The helper cell line stably expresses T7 RNA polymerase as well as measles virus N and P proteins by tricistronic mRNA. Materials and Methods: For rescue of measles virus minigenome a stable helper cell line by using tricistronic expression vector was developed which expressed T7 RNA polymerase as well as measles virus N and P proteins. To construct the tricistronic expression vector, T7 RNA polymerase gene was cloned after cytomegalovirus [CMV] promoter and measles virus N and P proteins were under control of IRES [internal ribosome entry site] sequences. Our results indicated that measles virus minigenome could be rescued in this constructed helper cell line. Through this system, the measles virus minigenome was rescued. Further studies are necessary to improve the rescue efficiency. This may be possible by replacing the CMV promoter with the T7 promoter

Effect of dexamethasone, insulin and egf on the myogenic potential on human endometrial stem cell

Human endometrium contains mesenchymal stem cells [eMSC] which have the ability to differentiate into three cell lineages and the potential in therapeutic applications. We hypothesize that using environmental induction in culture media such as dexamethasone. human recombinant insulin and human epidermal growth factor [hEGF] can differentiate endometrial stem cells into myoblast. These agents have a broad range of effects in myoblast differentiation in-vitro. We used immunohystochemistry analysis and RT -PCR to evaluate the presence of skeletal muscle - specific proteins some of which are expressed in the early stage of differentiation including myoD and Desmin which expressed at later stages of differentiation. In conclusion eMSC can differentiate in culture media which contains above mentioned factors and use for therapeutic purpose in muscular degenerative disease

[Molecular evaluation of the 2'-aminoglycoside nucleotidyltransferase gene in Escherichia coli isolates that produce hemolysin and are sensitive to mannose type I pili and P]

Aminoglycosides are highly potent, broad-spectrum antibiotics with many desirable properties for the treatment of life-threatening infections. Escherichia coli [E. coli] is the most common cause of urinary tract infection [UTI]. Antibiotic resistance has recently become prevalent. Enzymatic inactivation of aminoglycosides by aminoglycoside-modifying enzymes is the main mechanism of resistance to these antibiotics in E. coli. The main purpose of this research is to evaluate the presence of the 2'-aminoglycoside nucleotidyltransferase [ant[2"]-Ia] gene in E. coli isolates sensitive to mannose and hemolysin production. After collecting 276 E. coli isolates from patients that referred to Tehran Heart Center, we used the disk diffusion method to determine the resistance patterns of isolates toward Gentamicin, Tobramycin, Kanamycin, Amikacin and Netilmicin antibiotics according to the CLSI principles. We evaluated hemolysin production by assessing the ability of the isolates to grow on sheep and human blood agar media. Chromosomal DNA of the isolates was extracted using DNA extraction kits and PCR method used for the detection of the ant[2"]-Ia gene. In order to study mannose sensitivity we used human RBCs. Results obtained from antibiotic resistance determination tests showed that the highest rate of resistance was observed against tobramycin [24/63%]. Of those resistant, 6% could produce hemolysin in both sheep and human blood agar media. Mannose sensitivity was observed in 14% of isolates during agglutination. There were 24.63% of E. coli isolates resistant to Tobramycin, 23.18% resistant to kanamycin, 21.01% resistant to gentamicin, 6.15% resistant to netilmicin and 3.62% resistant to amikacin. ant[2"]-Ia gene was detected in 47.88% of E. coli isolated from urine. Due to the high prevalence of urinary tract infections caused by uropathogenic E. coli [UPEC] strains and the increasing rate of antibiotic resistance, periodic evaluations should be conducted for outbreaks of resistance in order to select the most suitable treatment to prevent routinely increasing antibiotic resistance

[Screening phage display library for isolation of human colon cancer cell-binding peptide ligands]

Recently, phage display libraries have received enormous attention for identification and isolation of pharmaceutical molecules with diagnostic and therapeutic properties. Peptide libraries are known as one of the most important and widely used types of phage display libraries. In the current study, we aimed to screen the Ph.D.[TM]-7 phage display peptide library through biopanning for the identification of human colon adenocarcinoma-binding peptide ligands. Three rounds of biopanning were performed on SW480 as the target cell and fibroblast [HF-SF-PI3], AGS, KYSE-30 and Huh-7 as control cells. The displayed peptide-encoding regions in the genome of SW480-binding phages obtained from the final round of panning were amplified by plaque-PCR and subsequently sequenced. Bioinformatic tools were used to determine the sequence of target cell-binding peptides and further characterization of these peptides. Biopanning of the phage library led to the enrichment of several peptides among which the peptide with sequence "HAMRAQP" was the most dominant. Bioinformatic analysis of the isolated peptides indicated that they are not target unrelated peptides [TUP]. The peptides, in particular those with the highest frequency, due to having the capability of specific binding to SW480 cells represent the potential for use in targeting of therapeutic genes and drugs to colon cancer cells

[Dendrosomal curcumin upregulates expression of the long noncoding RNA gene MEG3 in U87MG glioblastoma cells]

Glioblastoma is an invasive tumor of the central nervous system. Epigenetic therapy of cancer is potentially very useful in reversing some of cancer defects due to reversibility of epigenetic alterations. MEG3 is a tumor suppressor long non-coding RNA [lncRNA] that expresses in the majority of normal tissues. Methylation of the MEG3 promoter region elicits a decrease in its expression in glioblastoma cells. Bioactive nutrients including curcumin offer great potential in altering DNA methylation status. Herein, we aim to investigate the epigenetic-based role of dendrosomal-curcumin [DNC] in upregulation of MEG3 expression in glioblastoma cells. We evaluated DNC entrance to U87MG cells with the use of the fluorescent characteristics of curcumin. Next we performed the MTT assay to evaluate DNC and dendrosome effects on cell viability. The ability of DNC to boost expression of MEG3 in DNA methylation regulation was accomplished by a study of the relative expressions of MEG3 and DNA methylation regulator enzymes, DNA methyltransferases [DNMT1, DNMT3A and 3B] using semi-quantitative and quantitative PCR. We observed the entrance of DNC into U87MG cells. DNC significantly caused U87MG cell death in a time and dose-dependent manner. However dendrosome did not show any toxic effect on this cell line. Data acquired from gene expression assays determined that DNC upregulated MEG3 expression [P<0.05] and downregulated DNMT3B expression [P<0.05]. There was no significant effect on DNMT1, 3A expression in U87MG cells. The data showed that DNC could awaken epigenetically silenced tumor suppressor genes through an ambiguous route in glioblastoma cells. Notwithstanding, DNA hypomethylation has occurred by downregulation of DNMTs, inactive DNA demethylation and or active DNA demethylation, subsequently tumor suppressor genes such as MEG3 a cell growth regulator overexpressed. We concluded that DNC has useful characteristics in epigenetic therapy of glioblastoma

[Hsa-miR-133b expression profile during cardiac progenitor cell differentiation and its inhibitory effect on SRF expression]

Cardiac cell differentiation with the help of miRNAs has recently opened a promising window for the restoration of myocardial infarction. Independent miR-1-2/133a-1 and miR-206/133b clusters are known to be expressed in cardiac and skeletal muscles, respectively. miR-133b differs from miR-133a by only one nucleotide. The sequence similarity of these two miRNAs suggests that they target the same pathways and similar mRNA targets. The present study seeks to determine if miR-133b is expressed during the cardiac cell differentiation and if its expression is in reverse correlation with the SRF and CCND2 [as potential target genes] expression patterns. Human cardiac progenitor cells were prepared from Royan Stem Cell Bank [RSCB] and differentiated into cardiomyocytes. To initiate differentiation, cells were treated with 5-azacytidine as a demethylation factor. Then, ascorbic acid and TGFB1 were added every other day and twice per week, respectively. Differentiation into cardiomyocytes was confirmed by immunocytochemistry [ICC], flow cytometry and realtime PCR for some of the cardiac marker genes. The expression profiles of hsa-miR-133b and two of its potential target genes were also analyzed during the cardiac differentiation. Three weeks after the first differentiation induction, expression level of hsa-miR-133b was approximately five times higher than early stage expression [p<0.05]. During this process, the expression profile of SRF target gene was inversely correlated with hsamiR-133b expression. It is known that SRF is critically involved in the cell cycle. Considering increased miR-133b and decreased SRF expression levels during the late stages of heart cell differentiation, here we speculate that elevated expression of miR-133b blocks SRF expression and decreases cardiomyocytes proliferation in order to induce differentiation with direct targeting of SRF. Taken together, our data suggest that miR-133b along with miR-133a may be involved in cardiomyocytes differentiation.

[Dendrosomal curcumin induced apoptosis by suppression of pluripotency genes in 5637 bladder cancer cells]

The anti-cancer properties of curcumin, a poliphenol extract from the rhizome of curry, has been confirmed by many investigators. However, low levels of uptake, tissue distribution and rapid metabolism has limited its application as an anti-cancer drug. This study is aimed at increasing curcumin's water solubility due to a biodegradable, neutral and non-toxic micellar nano-carrier called dendrosome. This study intends to evaluate the role of dendrosomal-curcumin [DNC] in bladder cancer cell growth. We performed the MTT assay, flow cytometry and Annexin V-FLUOS [as an apoptosis detection kit] to evaluate cell death. The genetic mechanism of DNC-induced apoptosis was accomplished by a study of the relative expressions of OCT4A, OCT4B1, SOX-2 and Nanog using real-time PCR. DNC-induced cell death complied with a time and dose-dependent paradigm in the 5637 cell line. Cell cycle analysis revealed that the number of cells increased in pre-G1 and gradually decreased in G1 and S phases. This showed the inhibitory property of dendrosomal-curcumin on DNA synthesis. Data from real-time PCR determined that expressions of OCT4A, OCT4B1, SOX-2 and Nanog could be related to 5637 cancer cell growth. Dendrosomal-curcumin significantly suppressed mRNA expression of the above mentioned genes [p<0.01]. The data showed that DNC induced apoptosis by suppression of pluripotency genes in 5637 bladder cancer cells, which confirmed the useful characteristic of nano-drug in bladder cancer therapy.

Analysis of the effect of chronic morphine treatment on miRNA profile and introduction of the MAPK pathway as the target of differentially expressed miRNAS

Different signaling pathways have been identified that are involved in the cellular response to opiates. The mitogen-activated protein kinase pathway is one of the most important signaling pathways underlying the neuronal response to opiates. MicroRNAs [miRNAs] are considered to be post-transcriptional regulators of gene expression with paramount significance, which plays key roles in modulating cellular processes such as neuronal plasticity and synaptic consolidation. The purpose of this study is to identify miRNAs that are differentially expressed in response to chronic morphine treatment, and predict those genes that have a possible role in this process. Because the MAPK pathway in involved in morphine dependence and participates in hypersensitivity to pain, determining miRNAs that modulate this pathway could be insightful in morphine dependence treatment and pain control. In this study, the BE[2]-C neuroblastoma cell line was chronically treated with morphine sulphate and the changes in expression of 750 miRNAs were analyzed by real time PCR. Two up- and down- regulated groups of miRNAs were determined to be differentially expressed in response to morphine: i] has-mir-193a-3p, -212, -181c, -362-3p, -639, -646 and ii] has-mir-412, -937, -558, -552, -943, -628-5p, -593, -555, -636, -643, 566, -571, -642, -653, -611, -31, let7-g. The analysis of differentially expressed miRNAs showed that the MAPK signaling pathway could be regarded as a signaling pathway with utmost significance in chronic morphine response. Due to the role played by MAPK pathway in cellular response to morphine exposure, we can propose that protein phosphorylation has a presumable part in this response